US10722553B2 - Medicament containing recombinant mistletoe lectins for the treatment of brain tumors - Google Patents

Medicament containing recombinant mistletoe lectins for the treatment of brain tumors Download PDF

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US10722553B2
US10722553B2 US16/077,512 US201716077512A US10722553B2 US 10722553 B2 US10722553 B2 US 10722553B2 US 201716077512 A US201716077512 A US 201716077512A US 10722553 B2 US10722553 B2 US 10722553B2
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mistletoe lectin
mistletoe
cells
recombinant mistletoe
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Hans Lentzen
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Melema Pharma GmbH
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C07K14/42—Lectins, e.g. concanavalin, phytohaemagglutinin

Definitions

  • the invention relates to a medicament and/or pharmaceutical composition containing recombinant mistletoe lectins for the treatment of brain tumours, in particular primary brain tumours, gliomas, glioblastomas, meningiomas and pituitary adenomas, and use thereof.
  • Primary brain tumours such as gliomas, glioblastomas, meningiomas and pituitary adenomas, start from the neuroepithelium, ganglion cells, meninges, nerve sheaths, general nervous supporting tissue or neuroglia and pituitary or ectopic intracranial tissues (germ cell tumours or deformity tumours), and their causes are considered in particular to lie in genetic and hormonal factors, oncogenic viruses, and exogenous carcinogens.
  • tumours of the central nervous system localised in the brain, of varying differentiation and comprising various sub-types, such as: astrocytic tumours, oligodendrogliomas, mixed gliomas (oligoastrocytomas), ependymomas, tumours of the plexus choroideus, retinoblastomas, etc.
  • the WHO degree of classification is based on dignity: grade I (non-malignant, benign), grade II (semi-benign; postoperative life expectancy 3-5 years), grade III (semi-malignant; postoperative life expectancy 2-3 years), grade IV (malignant; postoperative life expectancy 6-16 months); and frequency: proportion of total or primary brain tumours in all tumour diseases: 7-9% (Kleihues, P., Louis, D. N., Scheithauer, B. W., Rorke, L. B., Reifenberger, G., Burger, P. C., and Cavenee, W. K. (2002) The WHO classification of tumors of the nervous system. J. Neuropathol. Exp. Neurol. 3, 215-225).
  • a glioma is characterised histologically in (giant cell) (oligo)astrocytoma, oligodendroglioma, mixed gliomas, glioblastoma, and is differentiated depending on growth, such as isomorphic, anaplastic, pilocytic, etc. Sub-groups of gliomas can also be predicted on the basis of the loss of heterozygosity (Smith, J. S., and Jenkins, R. B. (2000) Genetic alterations in adult diffuse glioma: occurrence, significance, and prognostic implications. Front Biosci.
  • tumour suppressor genes Tews, B., Felsberg, J., Hartmann, C., Kunitz, A., Hahn, M., Toedt, G., Mau, K., Hummerich, L., von Deimling, A., Reifenberger, G., and Lichter, P. (2006) Identification of novel oligodendroglioma-associated candidate tumor suppressor genes in 1p36 and 19q13 using microarray-based expression profiling. Int J Cancer. 119, 792-800).
  • GBM glioblastomas
  • NK Natural killer cells as part of the natural immune system play a key role in the destruction of cancer cells. GBM cells develop strategies for avoiding this killing by down-regulating proteins that are necessary for interaction with NK cells, these being known as danger/stranger protein major histocompatibility complex (MHC), MHC class I polypeptide-related sequence (MIC)-A and -B, or UL16 binding proteins (ULBP) 1, 2, 3, more specifically by means of TGF- ⁇ mediated immunosuppression.
  • MHC danger/stranger protein major histocompatibility complex
  • MIC MHC class I polypeptide-related sequence
  • ULBP UL16 binding proteins
  • TGF- ⁇ is an essential feature of GBM, and high concentrations of TGF- ⁇ can be detected in glioma patients in the cerebrospinal fluid, which is correlated with the growth of the tumour (Kjellman C, Olofsson S P, Hansson O et al: Expression of TGF-beta isoforms, TGF-beta receptors, and SMAD molecules at different stages of human glioma. Int J Cancer 2000; 89: 251-258).
  • TGF- ⁇ is responsible for the down-regulation of MHC expression, increasing the differentiation of na ⁇ ve cells in T-reg cells, blocking dendritic cell maturation, and inducing cell death of K and T-cells (Eisele G, Wischhusen J, Mittelbronn M et al.: TGF-beta and metalloproteinases differentially suppress NKG2D ligand surface expression on malignant glioma cells. Brain 2006; 129: 2416-2425, Platten M, Wick W, Weller M: Malignant glioma biology: role for TGF-beta in growth, motility, angiogenesis, and immune escape. Microsc Res Tech 2001; 52: 401-410).
  • brain tumours in particular primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas.
  • Mistletoe extracts have been used therapeutically for hundreds of years. In particular in cancer therapy, mistletoe preparations have been used with varying levels of success (Bocci V 1993 J Biol Regulators and Homeostatic Agents 7(1): 1-6; Gabius H-J, Gabius S, Joshi S S et al. 1993 Planta Med 60: 2-7; Gabius H-J & Gabius S 1994 PZ 139: 9-16; Ganguly C & Das S 1994 Chemotherapy 40: 272-278, Hajto T, Hostanska K, Gabius H_J 1989 Cancer Res 49: 4803-4808, Hajto T, Hostanska K, Frei K et al. 1990 Cancer Res. 50: 3322-3326).
  • mistletoe lectins viscumins, Viscum album Agglutinine, VAA.
  • mistletoe lectin besides a cytotoxic effect, also brings about non-specific immunostimulation, the positive effects of which are used for therapy in tumour patients.
  • Various studies with mistletoe lectin in vitro (Hajto et al., 1990 (supra); Mannel D N, Becker H, Gundt A et al. 1991 Cancer Immunol Immunother 33: 177-182; Beuth J, Ko K L, Tunggal L et al.
  • mistletoe lectins ML-I, ML-II, ML-III
  • the ML-I lectin consists of two glycosylated A- and B-chains (MLA and MLB).
  • the A-chain is responsible for enzymatic inactivation of ribosomes (Endo Y, Tsurugi K & Franz H 1988 FEBS Lett 231: 378-380), whereas the B-chain is involved in carbohydrate bonding.
  • the two chains are linked to one another by disulphide bridges.
  • the resultant mistletoe lectin monomers can clump together to form dimers, with formation of non-covalent bonds.
  • EP 0751221 describes the preparation of mistletoe lectin polypeptides in a pure state as a structurally homogenous substance, wherein, proceeding from the gene sequence of mistletoe lectin, recombinant, highly pure individual chains (A-chain, B-chain) are produced, which can be re-associated in vitro and thus provide a recombinant mistletoe lectin holoprotein, which is homogeneous in respect of its protein chemistry, enzymatically and structurally, also known as Aviscumine.
  • the recombinant mistletoe lectin polypeptide is suitable as holoprotein and as sub-chain and in the form of sub-fragments for therapeutic purposes and is included within the scope of the invention.
  • WO2012104355A1 also describes the antiviral effect of recombinant mistletoe lectins.
  • WO2012136857A1 discloses the treatment of skin cancer, in particular of a malignant melanoma also in the form of a metastasising tumour by means of recombinant mistletoe lectins.
  • recombinant mistletoe lectins were used advantageously in the treatment of tumour diseases.
  • Podlech et al (Podlech O, Harter P N, Mittelbronn M et al.: Fermented mistletoe extract as a multimodal antitumoral agent in gliomas. Evid Based Complement Alternat Med 2012: 501796) describes the use of ISCADOR—a fermentatively obtained mistletoe extract—as growth inhibitor for GBM and indicates the antitumoral suitability of ISCADOR for the treatment of GBM.
  • Lenartz (Lenartz et al., Immunoprotective Activity of the Galactoside-Specific lectin from mistletoe after Tumor Destructive Theraly in Glioma Patients, Anticancer Research 16: 3799-3802 (1996)) reports on a mistletoe extract (ML-1) for the treatment of glioma patients, wherein the mistletoe extracts have a specific glycosylation.
  • mistletoe lectin obtained from Korean mistletoe for example should be assigned to the RIP II proteins, but has significant structural differences in the structure and conformation compared to the recombinant mistletoe lectin discussed here (Kang T B, Song S K, Yoon T J et al. 2007 J Biochem Mol Biol 40(6): 959-965). It is particularly disadvantageous that no exact dose adjustment is possible and that mistletoe lectins obtained from plant-based fermentatively or non-fermentatively produced extracts comprise impurities.
  • the production of each new mistletoe extract batch yields a product that is not identical to the previous batch, with different contents of its ingredients including the glycosylated mistletoe lectins.
  • the invention relates to a medicament and/or pharmaceutical composition containing recombinant mistletoe lectins for the treatment of the brain tumours, in particular primary brain tumours, gliomas, glioblastomas, meningionas and pituitary adenomas, and use thereof.
  • FIG. 1 depicts a graph of the number of migrated cells as a function of incubation treatment of human gliomacells.
  • ISC Qu ISCADOR Q.
  • Avi Aviscumine.
  • ML-1 mistletoe lectin I.
  • Anti-M1 Ab mistletoe lectin antibody.
  • FIG. 2 depicts a comparative heat map of upregulated and downregulated genes as a function of treatment with ISCADOR Qu, Aviscumine, or ML-1.
  • the recombinant mistletoe lectins according to the invention advantageously do not have any such glycosylation, are absolutely pure, and can be produced reproducibly.
  • recombinant mistletoe lectins not only have the aforesaid advantages, such as improved reproducibility, homogeneity and adjustable dosing, but provide improved NK cell cytotoxicity compared to a plant-based or fermentative mistletoe extract from the prior art via the NK cell surface marker NKG2D and are therefore particularly suitable for the treatment of brain tumours, gliomas, glioblastomas, meningiomas and pituitary adenomas.
  • Recombinant mistletoe lectins also demonstrate a specific and advantageous anti-migratory effect on brain tumour cells.
  • NK cells Natural killer (NK) cells as constituents of the natural immune system play a key role in the destruction of cancer cells.
  • the docking of NK cells to tumour cells is necessary.
  • the NKG2d receptors on NK cells and for activation of necessary surface proteins (NKp30, NKp44, NKp46) play an important role.
  • the effect of the recombinant mistletoe lectins is compared with the effect of the fermented mistletoe extract ISCADOR Q on the interaction of NK cells with the GBM cell LNT0229-Luc.
  • the object of the present invention thus lies in providing a medicament and pharmaceutical agent for the treatment of brain tumours, in particular primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas.
  • brain tumours in particular primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas.
  • the object is achieved by providing a medicament and a pharmaceutical composition, wherein these contain recombinant mistletoe lectins for the treatment of brain tumours, in particular primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas.
  • brain tumours in particular primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas.
  • the medicament according to the invention preferably comprises the mistletoe lectin A chain (MLA) or the mistletoe lectin B chain (MLB), in each case individually or together, also in the form of dimers (see for example EP 0 751 221 or EP 1 051 495).
  • MVA mistletoe lectin A chain
  • MLB mistletoe lectin B chain
  • the recombinant mistletoe lectin polypeptide of the mistletoe lectin A chain comprises the following sequences: SEQ ID No. 1-3, inclusive of the isoforms thereof or a functional fragment thereof.
  • the recombinant mistletoe lectin polypeptide of the mistletoe lectin B chain comprises the following sequences: SEQ ID No. 4-12, inclusive of the isoforms thereof or a functional fragment thereof (hereinafter all “recombinant mistletoe lectins”).
  • a recombinant mistletoe lectin according to the invention is a heterodimer consisting of the sequences SEQ ID No. 1 and SEQ ID No. 4, see for example EP 0 751 221 (what is known as Aviscumine).
  • fragments or derivatives of the polypeptides induce the same signals in a cell as the aforesaid peptides.
  • fragments are peptide domains with defined functions.
  • the “same biological function” also comprises the cytotoxicity, immunostimulation (both of the native and adaptive immune system), stimulation of the expression or the activation of surface markers, the induction of apoptosis, or endorphin stimulation.
  • biological activity of the recombinant mistletoe lectin is understood here to mean any biological activity from the spectrum of all biological activities of the recombinant mistletoe lectin.
  • a function of this kind is for example the pharmacological effect of the recombinant mistletoe lectin.
  • the medicament according to the invention contains a recombinant mistletoe lectin polypeptide with the sequences SEQ ID No. 1-12 or a functional fragment thereof or any combination thereof.
  • mistletoe lectins takes effect in patient populations that do not respond to tumour preparations by means of standard therapy or that comprise non-responders or therapy failures.
  • the invention therefore comprises patients or patient populations of non-responders and therapy failures for the treatment of brain tumours, in particular primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas, in which standard tumour therapy is unsuccessful.
  • brain tumours in particular primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas, in which standard tumour therapy is unsuccessful.
  • brain tumour in accordance with the invention comprises primary brain tumours, such as gliomas, meningiomas and pituitary adenomas, starting from the neuroepithelium, ganglion cells, meninges, nerve sheaths, general nervous supporting tissue or neuroglia and pituitary or ectopic intracranial tissues (germ cell tumours or deformity tumours), and their causes are considered in particular to lie in genetic and hormonal factors, oncogenic viruses, and exogenous carcinogens.
  • primary brain tumours such as gliomas, meningiomas and pituitary adenomas, starting from the neuroepithelium, ganglion cells, meninges, nerve sheaths, general nervous supporting tissue or neuroglia and pituitary or ectopic intracranial tissues (germ cell tumours or deformity tumours), and their causes are considered in particular to lie in genetic and hormonal factors, oncogenic viruses, and exogenous carcinogens.
  • a preferred embodiment according to the invention is the treatment of gliomas (see for example the description of the indications in Pschyrembel®, 266 th edition 2014, De Gruyter Verlag, Berlin).
  • the invention additionally relates to a medicament for the treatment of brain tumours, in particular primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas, which contains the recombinant mistletoe lectin polypeptide optionally together with a pharmaceutically acceptable carrier, thus forming a pharmaceutical composition.
  • a pharmaceutically acceptable carrier examples include buffered saline solutions, water, etc., various kinds of detergents, sterile solutions, etc.
  • Medicaments that comprise such carriers can be formulated by means of known conventional methods. These medicaments can be administered to an individual in a suitable dose.
  • the administration can be implemented locally, orally or parenterally, for example intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, intranasally, intrabronchially or intradermally, or via a catheter at a point in an artery.
  • the kind of dosing if determined by the treating doctor in accordance with the clinical factors. It is known to a person skilled in the art that the kind of dosing is dependent on various factors, such as body size or weight, body surface, age, sex, or general health of the patient, but also on the specific agent to be administered, the duration and kind of administration, and any other medicaments that might be administered in parallel.
  • composition comprising the recombinant mistletoe polypeptides according to the invention can be administered locally or systemically.
  • a dosing of the mistletoe lectins according to the invention for human use of 2-10 ng/kg (body weight) has proven to be advantageous.
  • the administered amount is preferably 5 ng/kg body weight.
  • the preferred human dosing not based on body weight is 350 ng.
  • the medicament according to the invention is applied over a period of time of at least 8 weeks at a frequency of 1 ⁇ daily to 1 ⁇ weekly.
  • the medicament is preferably administered 2 to 3 ⁇ weekly, particularly preferably 2 ⁇ weekly.
  • the invention therefore relates to a method for dosing the recombinant mistletoe lectins according to the invention or the medicament according to the invention, wherein the dosing is 2 to 10 ng/kg (body weight).
  • the invention relates to a method for dosing the recombinant mistletoe lectins according to the invention or the medicament according to the invention, wherein the dose is 200-500 ng, in particular 350 ng, and is administered to the patient at least 1 ⁇ weekly.
  • LNT-229-Luc cells grow in DMEM (Sigma, Taufkirchen, Germany) medium with 10% foetal calf serum and penicillin/streptomycin in a humid atmosphere enriched with 5% CO2.
  • PBMC peripheral blood mononuclear cells
  • irradiated RPMI 8866 feeder cells ATCC, USA
  • RPMI medium Sigma, Taufmün, Germany
  • the NK cells are purified with the aid of an NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) >98%.
  • Their lytic activity in relation to GBM cells is determined via the Luciferase activity measurement.
  • the NK cells are pre-incubated with antibodies against NKG2D (BioLegend, Fell, Germany) for 30 minutes prior to their 4-hour co-cultivation (effector/target cell 20:1) with the GBM cells.
  • the GBM cells are in turn pre-treated for 24 h with Aviscumine or ISCADOR Q prior to the co-cultivation with the NK cells and are washed in the co-culture prior to their use.
  • Aviscumine influences the effect of NK cells on the GBM cell by intensifying the interaction of their receptor NKG2D with the target cell.
  • ISCADOR Q 200 mg is diluted 2000 times for incubation experiments (100 micrograms/ml).
  • the extract contains, inter alia, various mistletoe lectins, the content of which in the used batch is stated as 15.050 micrograms mistletoe lectin/ml.
  • the concentration of 7.5 nanogram lectins per ml leads after 48 h to an inhibition of growth of almost 20-30% in LNT-229 and SMA560 glioma cells.
  • ISCADOR Q inter alia also contains various cytotoxic viscotoxins, the content of which in the used batch is stated as 364 micrograms viscotoxin/ml. This means a viscotoxin concentration of 182 nanograms viscotoxins per ml incubation solution.
  • mistletoe lectin Aviscumine inhibits the growth of various brain tumour cells by 100% in a concentration range of 0.4-11 ng/ml and as an inhibitor of brain tumour cells is thus >10 times more potent than ISCADOR Q. If it is desired to attribute the effect of ISCADOR Q to the mistletoe lectins contained therein, mistletoe lectin-antagonising ingredients must thus be assumed in the fermented extract.
  • mistletoe extracts are also cytotoxic (see Eggenschwiler J, von B L, Stritt B, Pruntsch D, Ramos M, Urech K, Rist L, Simoes-Wust A P, Viviani A; Mistletoe lectin is not the only cytotoxic component in fermented preparations of Viscum album from white fir ( Abies pectinata ). BMC Complement Altern Med 2007, 7:14).
  • the human glioma cell line LNT-229 was incubated for 24 h with the mistletoe lectin-containing extract Iscador Qu, Aviscumine and native mistletoe lectin I.
  • the used concentrations corresponded to 8 ng ML-I/ml.
  • the cells were washed for removal of the active substances, and in each case 20,000 cells were applied in a cell migration chamber (Boyden Chamber) to the upper layer of a polycarbonate membrane, which on account of its 8 mm pores is permeable for the cells and which divides the chamber into an upper and lower compartment.
  • Cells that have migrated through the membrane either remain adhered to the underside of the membrane depending on their adherent properties (content of pro-migratory or anti-migratory proteins) or pass into the buffer located in the lower compartment.
  • the membrane can be removed, and the adhering cells stained and counted. The difference between cell count and adhering cells is the number of migrated cells. The results are shown in FIG. 1 .
  • the number of migrating cells reduces on account of the mistletoe lectin effect.
  • Antibodies against mistletoe lectin cancel the effect.

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Abstract

Methods of treating a brain tumor by administering a drug containing recombinant mistletoe lectin are described. The recombinant mistletoe lectin can be a mistletoe lectin A-chain, a mistletoe lectin B-chain, or a combination thereof. Brain tumors that can be treated by the method described include primary brain tumours, gliomas, glioblastomas, meningiomas and pituitary adenomas.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a national stage application (under 35 U.S.C. § 371) of PCT/EP2017/053429, filed Feb. 15, 2017, which claims benefit of European Application No. 16155735.0, filed Feb. 15, 2016, both of which are incorporated herein by reference in their entirety.
REFERENCE TO A SEQUENCE LISTING
The instant application contains a sequence listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 10, 2018, is named 219557_0001_00_US_580558_ST25, and is 32,792 bytes in size.
FIELD OF THE INVENTION
The invention relates to a medicament and/or pharmaceutical composition containing recombinant mistletoe lectins for the treatment of brain tumours, in particular primary brain tumours, gliomas, glioblastomas, meningiomas and pituitary adenomas, and use thereof.
BACKGROUND
Primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas, start from the neuroepithelium, ganglion cells, meninges, nerve sheaths, general nervous supporting tissue or neuroglia and pituitary or ectopic intracranial tissues (germ cell tumours or deformity tumours), and their causes are considered in particular to lie in genetic and hormonal factors, oncogenic viruses, and exogenous carcinogens. They are actual tumours of the central nervous system (CNS) localised in the brain, of varying differentiation and comprising various sub-types, such as: astrocytic tumours, oligodendrogliomas, mixed gliomas (oligoastrocytomas), ependymomas, tumours of the plexus choroideus, retinoblastomas, etc.
The WHO degree of classification is based on dignity: grade I (non-malignant, benign), grade II (semi-benign; postoperative life expectancy 3-5 years), grade III (semi-malignant; postoperative life expectancy 2-3 years), grade IV (malignant; postoperative life expectancy 6-16 months); and frequency: proportion of total or primary brain tumours in all tumour diseases: 7-9% (Kleihues, P., Louis, D. N., Scheithauer, B. W., Rorke, L. B., Reifenberger, G., Burger, P. C., and Cavenee, W. K. (2002) The WHO classification of tumors of the nervous system. J. Neuropathol. Exp. Neurol. 3, 215-225).
A glioma is characterised histologically in (giant cell) (oligo)astrocytoma, oligodendroglioma, mixed gliomas, glioblastoma, and is differentiated depending on growth, such as isomorphic, anaplastic, pilocytic, etc. Sub-groups of gliomas can also be predicted on the basis of the loss of heterozygosity (Smith, J. S., and Jenkins, R. B. (2000) Genetic alterations in adult diffuse glioma: occurrence, significance, and prognostic implications. Front Biosci. 5, 213-231), which lead to a loss of tumour suppressor genes (Tews, B., Felsberg, J., Hartmann, C., Kunitz, A., Hahn, M., Toedt, G., Neben, K., Hummerich, L., von Deimling, A., Reifenberger, G., and Lichter, P. (2006) Identification of novel oligodendroglioma-associated candidate tumor suppressor genes in 1p36 and 19q13 using microarray-based expression profiling. Int J Cancer. 119, 792-800).
In particular, glioblastomas (GBM) are one of the most aggressive brain tumours. The median survival of GBM patients, even with the best therapy preconditions, is only approximately 12-15 months. Natural killer (NK) cells as part of the natural immune system play a key role in the destruction of cancer cells. GBM cells develop strategies for avoiding this killing by down-regulating proteins that are necessary for interaction with NK cells, these being known as danger/stranger protein major histocompatibility complex (MHC), MHC class I polypeptide-related sequence (MIC)-A and -B, or UL16 binding proteins (ULBP) 1, 2, 3, more specifically by means of TGF-β mediated immunosuppression. To this end the overexpression of TGF-β is an essential feature of GBM, and high concentrations of TGF-β can be detected in glioma patients in the cerebrospinal fluid, which is correlated with the growth of the tumour (Kjellman C, Olofsson S P, Hansson O et al: Expression of TGF-beta isoforms, TGF-beta receptors, and SMAD molecules at different stages of human glioma. Int J Cancer 2000; 89: 251-258). In addition, TGF-β is responsible for the down-regulation of MHC expression, increasing the differentiation of naïve cells in T-reg cells, blocking dendritic cell maturation, and inducing cell death of K and T-cells (Eisele G, Wischhusen J, Mittelbronn M et al.: TGF-beta and metalloproteinases differentially suppress NKG2D ligand surface expression on malignant glioma cells. Brain 2006; 129: 2416-2425, Platten M, Wick W, Weller M: Malignant glioma biology: role for TGF-beta in growth, motility, angiogenesis, and immune escape. Microsc Res Tech 2001; 52: 401-410).
There is thus a great need to provide medicaments for the treatment and prophylaxis of brain tumours, in particular primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas.
Mistletoe extracts have been used therapeutically for hundreds of years. In particular in cancer therapy, mistletoe preparations have been used with varying levels of success (Bocci V 1993 J Biol Regulators and Homeostatic Agents 7(1): 1-6; Gabius H-J, Gabius S, Joshi S S et al. 1993 Planta Med 60: 2-7; Gabius H-J & Gabius S 1994 PZ 139: 9-16; Ganguly C & Das S 1994 Chemotherapy 40: 272-278, Hajto T, Hostanska K, Gabius H_J 1989 Cancer Res 49: 4803-4808, Hajto T, Hostanska K, Frei K et al. 1990 Cancer Res. 50: 3322-3326). It has been found that the therapeutic effects are conveyed in particular by what are known as mistletoe lectins (viscumins, Viscum album Agglutinine, VAA). Mistletoe lectin, besides a cytotoxic effect, also brings about non-specific immunostimulation, the positive effects of which are used for therapy in tumour patients. Various studies with mistletoe lectin in vitro (Hajto et al., 1990 (supra); Mannel D N, Becker H, Gundt A et al. 1991 Cancer Immunol Immunother 33: 177-182; Beuth J, Ko K L, Tunggal L et al. 1993 Drug Res 43: 166-169) and in vivo (Hajto T 1986 Oncology 43 suppl 1: 51-65; Hajto et al., 1989 (supra), Beuth J, Ko H L, Gabius H-J et al. 1991 In Vivo 5: 29-32; Beuth J, Ko H L, Gabius H-J et al. 1992 J Clin Invest 70: 658-661), and clinical studies (Beuth et al., 1992 (supra)) have demonstrated an increased release of inflammatory cytokines (TNF-alpha, IL-1, IL-6) and an activation of cellular components of the immune system (TH-cells, NK-cells, B- and T-lymphocytes) (Braedel-Ruoff S: Immunomodulatory effects of Viscum album extracts on natural killer cells: review of clinical trials. Forsch Komplementmed 2010; 17: 63-73, Gren A: Effects of Iscador preparations on the reactivity of mouse immune system. Neuro Endocrinol Lett 2009; 30: 530-534, Lee C H, Kim J K, Kim H Y et al.: Immunomodulating effects of Korean mistletoe lectin in vitro and in vivo. Int Immunopharmacol 2009; 9: 1555-1561, Nikolai G, Friedl P, Werner M et al: Effect of a mistletoe extract (Iscador QuFrF) on viability and migratory behavior of human peripheral CD4+ and CD8+ T lymphocytes in three-dimensional collagen lattices. In Vitro Cell Dev Biol Anim 1997; 33: 710-716).
By analysis of the mistletoe extract, it has been possible thus far to identify three mistletoe lectins (ML-I, ML-II, ML-III) having different molecular weights and sugar-binding specificities. It has been found that the immunostimulating effect of the mistletoe extract on can be attributed to ML-I. The ML-I lectin consists of two glycosylated A- and B-chains (MLA and MLB). The A-chain is responsible for enzymatic inactivation of ribosomes (Endo Y, Tsurugi K & Franz H 1988 FEBS Lett 231: 378-380), whereas the B-chain is involved in carbohydrate bonding. The two chains are linked to one another by disulphide bridges. The resultant mistletoe lectin monomers can clump together to form dimers, with formation of non-covalent bonds.
It is possible to produce the biologically active mistletoe lectin advantageously recombinantly. EP 0751221 describes the preparation of mistletoe lectin polypeptides in a pure state as a structurally homogenous substance, wherein, proceeding from the gene sequence of mistletoe lectin, recombinant, highly pure individual chains (A-chain, B-chain) are produced, which can be re-associated in vitro and thus provide a recombinant mistletoe lectin holoprotein, which is homogeneous in respect of its protein chemistry, enzymatically and structurally, also known as Aviscumine. According to EP 0751221 the recombinant mistletoe lectin polypeptide is suitable as holoprotein and as sub-chain and in the form of sub-fragments for therapeutic purposes and is included within the scope of the invention.
Document WO2012104355A1 also describes the antiviral effect of recombinant mistletoe lectins. WO2012136857A1 discloses the treatment of skin cancer, in particular of a malignant melanoma also in the form of a metastasising tumour by means of recombinant mistletoe lectins.
Previously, recombinant mistletoe lectins were used advantageously in the treatment of tumour diseases. The use of recombinant mistletoe lectins for the treatment of brain tumours however, in particular primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas, is not described in the prior art.
In the prior art, Podlech et al (Podlech O, Harter P N, Mittelbronn M et al.: Fermented mistletoe extract as a multimodal antitumoral agent in gliomas. Evid Based Complement Alternat Med 2012: 501796) describes the use of ISCADOR—a fermentatively obtained mistletoe extract—as growth inhibitor for GBM and indicates the antitumoral suitability of ISCADOR for the treatment of GBM.
Lenartz (Lenartz et al., Immunoprotective Activity of the Galactoside-Specific lectin from mistletoe after Tumor Destructive Theraly in Glioma Patients, Anticancer Research 16: 3799-3802 (1996)) reports on a mistletoe extract (ML-1) for the treatment of glioma patients, wherein the mistletoe extracts have a specific glycosylation.
The plant-based mistletoe lectins described in the prior art—whether or not obtained from fermentatively or non-fermentatively produced mistletoe extracts—are inhomogeneous (Soler M H, Stoeva S, Schwamborn C et al. 1996 FEBS Letter 399: 153-157, Soler H S, Stoeva S, Voelter W 1998 Biochem Biophys Res Comm 246: 596-601) and differ from one another non-uniformly in respect of their effect (EP 1051495 B1), and are not effective per se as active substance or as immunomodulator. Thus, the mistletoe lectin obtained from Korean mistletoe (Viscum album colaratum) for example should be assigned to the RIP II proteins, but has significant structural differences in the structure and conformation compared to the recombinant mistletoe lectin discussed here (Kang T B, Song S K, Yoon T J et al. 2007 J Biochem Mol Biol 40(6): 959-965). It is particularly disadvantageous that no exact dose adjustment is possible and that mistletoe lectins obtained from plant-based fermentatively or non-fermentatively produced extracts comprise impurities. Furthermore, the mistletoe lectins obtained from plant extracts—produced fermentatively or non-fermentatively—present differences in the glycosylation which influence the efficacy (in particular kinetics, etc.). Apart from this, the production of each new mistletoe extract batch yields a product that is not identical to the previous batch, with different contents of its ingredients including the glycosylated mistletoe lectins.
BRIEF SUMMARY OF THE INVENTION
The invention relates to a medicament and/or pharmaceutical composition containing recombinant mistletoe lectins for the treatment of the brain tumours, in particular primary brain tumours, gliomas, glioblastomas, meningionas and pituitary adenomas, and use thereof.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts a graph of the number of migrated cells as a function of incubation treatment of human gliomacells. ISC Qu: ISCADOR Q. Avi: Aviscumine. ML-1: mistletoe lectin I. Anti-M1 Ab: mistletoe lectin antibody.
FIG. 2 depicts a comparative heat map of upregulated and downregulated genes as a function of treatment with ISCADOR Qu, Aviscumine, or ML-1.
 DETAILED DESCRIPTION
The recombinant mistletoe lectins according to the invention advantageously do not have any such glycosylation, are absolutely pure, and can be produced reproducibly.
Surprisingly, it could now be found that recombinant mistletoe lectins not only have the aforesaid advantages, such as improved reproducibility, homogeneity and adjustable dosing, but provide improved NK cell cytotoxicity compared to a plant-based or fermentative mistletoe extract from the prior art via the NK cell surface marker NKG2D and are therefore particularly suitable for the treatment of brain tumours, gliomas, glioblastomas, meningiomas and pituitary adenomas. Recombinant mistletoe lectins also demonstrate a specific and advantageous anti-migratory effect on brain tumour cells.
Natural killer (NK) cells as constituents of the natural immune system play a key role in the destruction of cancer cells. In order to achieve destruction of tumour cells by NK cells, the docking of NK cells to tumour cells is necessary. Here, the NKG2d receptors on NK cells and for activation of necessary surface proteins (NKp30, NKp44, NKp46) play an important role. In the examples, the effect of the recombinant mistletoe lectins is compared with the effect of the fermented mistletoe extract ISCADOR Q on the interaction of NK cells with the GBM cell LNT0229-Luc.
The object of the present invention thus lies in providing a medicament and pharmaceutical agent for the treatment of brain tumours, in particular primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas.
The object is achieved by providing a medicament and a pharmaceutical composition, wherein these contain recombinant mistletoe lectins for the treatment of brain tumours, in particular primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas.
The medicament according to the invention preferably comprises the mistletoe lectin A chain (MLA) or the mistletoe lectin B chain (MLB), in each case individually or together, also in the form of dimers (see for example EP 0 751 221 or EP 1 051 495).
The recombinant mistletoe lectin polypeptide of the mistletoe lectin A chain comprises the following sequences: SEQ ID No. 1-3, inclusive of the isoforms thereof or a functional fragment thereof.
The recombinant mistletoe lectin polypeptide of the mistletoe lectin B chain comprises the following sequences: SEQ ID No. 4-12, inclusive of the isoforms thereof or a functional fragment thereof (hereinafter all “recombinant mistletoe lectins”).
More preferably, a recombinant mistletoe lectin according to the invention is a heterodimer consisting of the sequences SEQ ID No. 1 and SEQ ID No. 4, see for example EP 0 751 221 (what is known as Aviscumine).
The term “functional fragment” in conjunction with this invention defines fragments or the aforesaid polypeptides which have the same biological function as the polypeptide presented above with the corresponding amino acid sequence.
The term “same biological function” in this context for example describes the fact that fragments or derivatives of the polypeptides induce the same signals in a cell as the aforesaid peptides. Examples of fragments are peptide domains with defined functions. The “same biological function” also comprises the cytotoxicity, immunostimulation (both of the native and adaptive immune system), stimulation of the expression or the activation of surface markers, the induction of apoptosis, or endorphin stimulation.
The term “biological activity of the recombinant mistletoe lectin” is understood here to mean any biological activity from the spectrum of all biological activities of the recombinant mistletoe lectin. A function of this kind is for example the pharmacological effect of the recombinant mistletoe lectin.
Studies of ML-I monomers have revealed 25 isoforms, which can be attributed to different combinations of various A- and B-chains and different states of glycosylation of the chains.
For the present invention, a mistletoe lectin polypeptide or a fragment thereof, comprising the sequence variability of the various MLA and MLB chains, with sequences SEQ ID No. 1-12 is considered in accordance with the invention.
The medicament according to the invention contains a recombinant mistletoe lectin polypeptide with the sequences SEQ ID No. 1-12 or a functional fragment thereof or any combination thereof.
It is also preferred that the use of recombinant mistletoe lectins according to the invention takes effect in patient populations that do not respond to tumour preparations by means of standard therapy or that comprise non-responders or therapy failures.
The invention therefore comprises patients or patient populations of non-responders and therapy failures for the treatment of brain tumours, in particular primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas, in which standard tumour therapy is unsuccessful.
The term “brain tumour” in accordance with the invention comprises primary brain tumours, such as gliomas, meningiomas and pituitary adenomas, starting from the neuroepithelium, ganglion cells, meninges, nerve sheaths, general nervous supporting tissue or neuroglia and pituitary or ectopic intracranial tissues (germ cell tumours or deformity tumours), and their causes are considered in particular to lie in genetic and hormonal factors, oncogenic viruses, and exogenous carcinogens.
A preferred embodiment according to the invention, however, is the treatment of gliomas (see for example the description of the indications in Pschyrembel®, 266th edition 2014, De Gruyter Verlag, Berlin).
The invention additionally relates to a medicament for the treatment of brain tumours, in particular primary brain tumours, such as gliomas, glioblastomas, meningiomas and pituitary adenomas, which contains the recombinant mistletoe lectin polypeptide optionally together with a pharmaceutically acceptable carrier, thus forming a pharmaceutical composition. Examples of particularly suitable pharmacologically acceptable carriers are known to a person skilled in the art of tumour medicine and comprise buffered saline solutions, water, etc., various kinds of detergents, sterile solutions, etc. Medicaments that comprise such carriers can be formulated by means of known conventional methods. These medicaments can be administered to an individual in a suitable dose. The administration can be implemented locally, orally or parenterally, for example intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, intranasally, intrabronchially or intradermally, or via a catheter at a point in an artery. The kind of dosing if determined by the treating doctor in accordance with the clinical factors. It is known to a person skilled in the art that the kind of dosing is dependent on various factors, such as body size or weight, body surface, age, sex, or general health of the patient, but also on the specific agent to be administered, the duration and kind of administration, and any other medicaments that might be administered in parallel.
The pharmaceutical composition comprising the recombinant mistletoe polypeptides according to the invention can be administered locally or systemically.
A dosing of the mistletoe lectins according to the invention for human use of 2-10 ng/kg (body weight) has proven to be advantageous. The dosing particularly advantageously lies in a range of 3-7 ng/kg. The administered amount is preferably 5 ng/kg body weight. The preferred human dosing not based on body weight is 350 ng.
The medicament according to the invention is applied over a period of time of at least 8 weeks at a frequency of 1× daily to 1× weekly. The medicament is preferably administered 2 to 3× weekly, particularly preferably 2× weekly.
The invention therefore relates to a method for dosing the recombinant mistletoe lectins according to the invention or the medicament according to the invention, wherein the dosing is 2 to 10 ng/kg (body weight). In particular, the invention relates to a method for dosing the recombinant mistletoe lectins according to the invention or the medicament according to the invention, wherein the dose is 200-500 ng, in particular 350 ng, and is administered to the patient at least 1× weekly.
The following examples and drawings serve to explain the invention, but the invention is not limited to these examples.
EXAMPLES AND DRAWINGS Example 1 of a Composition of the Medicament
Solution for injection: 1 mL ampoule with 0.5 mL to 1.0 mL injection solution
Aviscumine 200-500 ng
Sodium monohydrogen phosphate dihydrate 2.8 mg-5.6 mg
Sodium dihydrogen phosphate dihydrate 0.078 mg-0.155 mg
Sodium chloride 3.3 mg-6.7 mg
Polyoxyethylene sorbitan ester (polysorbate) 0.1 mg
Glutaminic acid 0.1 mg
Water for injection ad 0.5 ml to ad 1.0 mL
Example 2 of a Composition of the Medicament
Powder for preparation of a solution or injection, 2R glass vial with
Aviscumine 200-500 ng
Trehalose 40.0 mg
Sodium chloride 1.0 mg
Tris(hydroxymethyl)aminomethane (TRIS) 0.6 mg
Polyoxyethylene sorbitan ester (polysorbate) 0.1 mg
Hydrochloric acid to adjust the pH value for administration, the powder is dissolved with 0.5 mL or 1.0 mL water for injection.
Example 3
Method: LNT-229-Luc cells grow in DMEM (Sigma, Taufkirchen, Germany) medium with 10% foetal calf serum and penicillin/streptomycin in a humid atmosphere enriched with 5% CO2.
For isolation of the NK cells, PBMC was taken from the blood of healthy donors and co-cultivated with irradiated RPMI 8866 feeder cells (ATCC, USA) in RPMI medium (Sigma, Taufkirchen, Germany) in order to obtain polyclonal NK cell populations. The NK cells are purified with the aid of an NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) >98%. Their lytic activity in relation to GBM cells is determined via the Luciferase activity measurement. For the inactivation of the NK cell activity, the NK cells are pre-incubated with antibodies against NKG2D (BioLegend, Fell, Germany) for 30 minutes prior to their 4-hour co-cultivation (effector/target cell 20:1) with the GBM cells. The GBM cells are in turn pre-treated for 24 h with Aviscumine or ISCADOR Q prior to the co-cultivation with the NK cells and are washed in the co-culture prior to their use.
Result: Aviscumine influences the effect of NK cells on the GBM cell by intensifying the interaction of their receptor NKG2D with the target cell.
The blocking of the NKG2D receptor by antibody leads, as expected, to a reduction of the lysis conveyed by NK cells by 52% in the control group (medium); Aviscumine antagonises the effect of the antibody in part: The lysis conveyed by NK cells is therefore reduced only by 38% by the antibody. A comparison with the fermented mistletoe extract ISCADOR Q was not possible under these conditions, since with the same mistletoe lectin-based concentration of 8 ng/ml, the GBM cells without influence of NK cells are lysed to a much greater extent than conveyed by the control. Clearly, further ingredients of the fermented extract are directly cytotoxic (Table).
GBM cell lysis
Without antibody anti-NKG2D pre-
treatment treatment
Medium  67% 32.1% P < 0.05
(Inhibition:
d. lysis by 52.1%)
Aviscumine 68.4% 42.6% P < 0.05
(Inhibition:
d. lysis by 37.7%)
Iscador Q 83.2% 61.6% not
significant
According to Podlech, ISCADOR Q (200 mg) is diluted 2000 times for incubation experiments (100 micrograms/ml). The extract contains, inter alia, various mistletoe lectins, the content of which in the used batch is stated as 15.050 micrograms mistletoe lectin/ml. This means a mistletoe lectin concentration of 7.5 nanograms lectin per ml in the incubation experiments. The concentration of 7.5 nanogram lectins per ml leads after 48 h to an inhibition of growth of almost 20-30% in LNT-229 and SMA560 glioma cells.
ISCADOR Q inter alia also contains various cytotoxic viscotoxins, the content of which in the used batch is stated as 364 micrograms viscotoxin/ml. This means a viscotoxin concentration of 182 nanograms viscotoxins per ml incubation solution.
When using ISCADOR it is therefore unclear which of the numerous ingredients is responsible for the inhibition of growth of LNT-229 and SMA560 glioma cells.
The recombinant mistletoe lectin Aviscumine inhibits the growth of various brain tumour cells by 100% in a concentration range of 0.4-11 ng/ml and as an inhibitor of brain tumour cells is thus >10 times more potent than ISCADOR Q. If it is desired to attribute the effect of ISCADOR Q to the mistletoe lectins contained therein, mistletoe lectin-antagonising ingredients must thus be assumed in the fermented extract. Other fluctuating constituents of mistletoe extracts are also cytotoxic (see Eggenschwiler J, von B L, Stritt B, Pruntsch D, Ramos M, Urech K, Rist L, Simoes-Wust A P, Viviani A; Mistletoe lectin is not the only cytotoxic component in fermented preparations of Viscum album from white fir (Abies pectinata). BMC Complement Altern Med 2007, 7:14).
Example 4
The human glioma cell line LNT-229 was incubated for 24 h with the mistletoe lectin-containing extract Iscador Qu, Aviscumine and native mistletoe lectin I. The used concentrations corresponded to 8 ng ML-I/ml. After 24 h the cells were washed for removal of the active substances, and in each case 20,000 cells were applied in a cell migration chamber (Boyden Chamber) to the upper layer of a polycarbonate membrane, which on account of its 8 mm pores is permeable for the cells and which divides the chamber into an upper and lower compartment. Cells that have migrated through the membrane either remain adhered to the underside of the membrane depending on their adherent properties (content of pro-migratory or anti-migratory proteins) or pass into the buffer located in the lower compartment.
At the end of the experiment the membrane can be removed, and the adhering cells stained and counted. The difference between cell count and adhering cells is the number of migrated cells. The results are shown in FIG. 1.
Compared to an untreated control group, the number of migrating cells reduces on account of the mistletoe lectin effect. Antibodies against mistletoe lectin cancel the effect.
The strongest anti-migratory effect is attained by Aviscumine (supra) (p<0.001).
This data, therefore that Aviscumine presents the strongest effect, can be explained by the specific downregulation of the pro-migratory genes MTA1 and MTA2 and the upregulation of the anti-migratory genes BRMS1 and SERPINB5. These genes, in contrast to the other mistletoe lectin preparations, can be downregulated and upregulated only by Aviscumine (see comparative heat map in FIG. 2).

Claims (11)

The invention claimed is:
1. A method of treating a brain tumor comprising administering to a human patient with a brain tumor a drug containing recombinant mistletoe lectin, wherein the recombinant mistletoe lectin is selected from the group of amino acid sequences SEQ ID No. 1-12 or a combination thereof.
2. The method of claim 1, wherein the recombinant mistletoe lectin polypeptide is a mistletoe lectin A, selected from the group of amino acid sequences SEQ ID No. 1-3 or a combination thereof.
3. The method of claim 1, wherein the recombinant mistletoe lectin polypeptide is a mistletoe lectin B, selected from the group of amino acid sequences SEQ ID No. 4-12 or a combination thereof.
4. The method of claim 1, wherein the human patient is a non-responder to standard tumor therapy or has had a therapeutic failure of standard tumor therapy.
5. The method of claim 1, wherein the medicament is combined with at least one of a pharmaceutically acceptable carrier or further auxiliaries and additives.
6. The method of claim 1, wherein the drug is administered to the human patient in a dosage in a range of 3-7 ng recombinant mistletoe lectin per kg of body weight.
7. The method of claim 6, wherein the drug is administered in a dosage of 5 ng recombinant mistletoe lectin per kg of body weight.
8. The method of claim 1, wherein the drug is administered to the human patient in a dosage of 200-500 ng recombinant mistletoe lectin regardless of body weight of the human patient.
9. The method of claim 8, wherein the dosage of recombinant mistletoe lectin is 350 ng regardless of body weight of the human patient.
10. The method of claim 1, wherein the medicament is administered at least once a week, at least twice a week, or at least three times per week.
11. The method of claim 1, wherein the brain tumor is selected from the group consisting of a primary brain tumor, a glioma, a glioblastoma, a meningioma, and a pituitary adenoma.
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